Fig. 1: MCR-1 reverses the rough phenotype and induces surface polysaccharide synthesis in E. coli.

a Venn diagram showing differentially expressed genes (DEGs; fold change ≥2 or ≤−2, P < 0.05) comparing wild type (WT) vs. transconjugant (TC) and TCΔmcr-1 vs. TC based on RNA-seq analysis. The overlapping region represents mcr-1-regulated genes. b Number of mcr-1-regulated DEGs in WT (fold change ≥2 or ≤−2, P < 0.05). Red and blue indicate upregulated and downregulated genes, respectively. Gray indicates unchanged genes. c Clusters of Orthologous Groups (COG) classification of mcr-1-regulated DEGs in WT. Red and blue bars indicate upregulated and downregulated genes, respectively. d Heatmap of gene expression related to enterobacterial common antigen (ECA) synthesis in WT, TC, and TCΔmcr-1 mutant. Each column represents one biological replicate. Red indicates increased expression, and blue indicates decreased expression. e Surface polysaccharide profiles of WT (lane 1), TC (lane 2), TCΔmcr-1 mutant (lane 3), mcr-1-complemented strain (lane 4), and WT carrying the mcr-1 gene alone (lane 5). “+” indicates reintroduction of the mcr-1 gene via pACYC184. f Quantification of glycan levels by wheat germ agglutinin (WGA) staining, expressed relative to WT. Data are presented as means ± SD from n = 4 biologically independent replicates. P values were determined using one-way ANOVA with Tukey’s multiple comparison test. WT vs. TC, P < 0.0001; TC vs. TCΔmcr-1 mutant, P < 0.0001; TCΔmcr-1 mutant vs. TCΔmcr-1 mutant+pmcr-1, P < 0.0001; WT vs. WT+pmcr-1, P = 0.9415. ns not significant, ****P < 0.0001. g Transmission electron microscopy (TEM) analysis showing surface polysaccharide layer at the cell surface. Cells were stained with 1.5% phosphotungstic acid for 2 min. The experiments were repeated three times with similar results, and representative data from one of the experiments are shown (e, g). All transcriptomic data presented in a–d were obtained from the same RNA-seq dataset. The strains used were E. coli 15734 (WT), WT carrying pFORC82_3 (TC), TC harboring mcr-1-deleted pFORC82_3 (TCΔmcr-1), TC harboring mcr-1-deleted pFORC82_3 and pACYC184-mcr-1 (TCΔmcr-1 + pmcr-1), and WT harboring pACYC184-mcr-1 (WT + pmcr-1). Source data are provided as a Source Data file.